Mutations in the p53 tumor suppressor gene are the most frequent genetic alteration in cancer and are often associated with progression from benign to invasive stages with metastatic potential. Mutations inactivate tumor suppression by p53, and some endow the protein with novel gain of function (GOF) properties that actively promote tumor progression and metastasis. By comparative gene expression profiling of p53-mutated and p53-depleted cancer cells, we identified ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5) as a mutant p53 target gene, which functions as a uridine 5′-diphosphatase (UDPase) in the endoplasmic reticulum (ER) to promote the folding of N-glycosylated membrane proteins. A comprehensive pan-cancer analysis revealed a highly significant correlation between p53 GOF mutations and ENTPD5 expression. Mechanistically, mutp53 is recruited by Sp1 to the ENTPD5 core promoter to induce its expression. We show ENTPD5 to be a mediator of mutant p53 GOF activity in clonogenic growth, architectural tissue remodeling, migration, invasion, and lung colonization in an experimental metastasis mouse model. Our study reveals folding of N-glycosylated membrane proteins in the ER as a mechanism underlying the metastatic progression of tumors with mutp53 that could provide new possibilities for cancer treatment.M utations in the TP53 tumor suppressor gene are the most frequent genetic alterations in human cancer and commonly compromise the gene's tumor suppressor activity. p53-knockout mice succumb to tumors very early in life, arguing that the loss of function associated with p53 mutations is sufficient on its own to explain the high mutation frequency observed in patients with cancer (1). However, in striking contrast to mutations in other tumor suppressor genes, the vast majority of TP53 gene alterations in patients with cancer neither ablate p53 expression nor produce unstable or truncated proteins. Instead, p53 mutations are mostly missense mutations resulting in expression of mutant p53 (mutp53) proteins with only single-amino acid substitutions that accumulate to steady-state levels greatly exceeding those of wild-type p53 (wtp53) in normal tissues. Immunohistochemical positivity for p53 is therefore considered a diagnostic marker for the presence of a TP53 mutation (2). The high prevalence of missense mutations suggests a selective advantage during cancer progression, so it was hypothesized early on in p53 research that p53 mutations are neomorphic and endow the mutp53 protein with novel oncogenic functions that actively promote cancer progression and therapy resistance (2). These oncogenic properties are generally referred to as the mutp53 gain of function (GOF).Over the years, substantial experimental evidence for mutp53 GOF has accumulated (3-5). For example, mice expressing cancer-associated p53 hot spot mutations from the endogenous Trp53 gene locus are remarkably different from p53-deficient mice: tumorigenesis is accelerated, and the spectrum of tumors is shifted toward carcinomas and more meta...

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Tumours are heterogeneous cell populations that undergo clonal evolution during tumour progression, metastasis and response to therapy. Short hairpin RNAs (shRNAs) generate stable loss-of-function phenotypes and are versatile experimental tools to explore the contribution of individual genetic alterations to clonal evolution. In these experiments tumour cells carrying shRNAs are commonly tracked with fluorescent reporters. While this works well for cell culture studies and leukaemia mouse models, fluorescent reporters are poorly suited for animals with solid tumours—the most common tumour types in cancer patients. Here we develop a toolkit that uses secreted luciferases to track the fate of two different shRNA-expressing tumour cell clones competitively, both in vitro and in vivo. We demonstrate that secreted luciferase activities can be measured robustly in the blood stream of tumour-bearing mice to accurately quantify, in a minimally invasive manner, the dynamic evolution of two genetically distinct tumour subclones in preclinical mouse models of tumour development, metastasis and therapy.

Recent structural analysis of Fe-S centers in replication proteins and insights into the structure and function of DNA polymerase δ (DNA Pol δ) subunits have shed light on the key role played by this polymerase at replication forks under stress. The sequencing of cancer genomes reveals multiple point mutations that compromise the activity of POLD1, the DNA Pol δ catalytic subunit, whereas the loci encoding the accessory subunits POLD2 and POLD3 are amplified in a very high proportion of human tumors. Consistently, DNA Pol δ is key for the survival of replication stress and is involved in multiple long-patch repair pathways. Synthetic lethality arises from compromising the function and availability of the noncatalytic subunits of DNA Pol δ under conditions of replication stress, opening the door to novel therapies. Highlights DNA polymerase δ is key to surviving replication stress, and is found in almost every oncogene-transformed mammalian cell line. Cancer-linked point mutations are found in POLD1, whereas POLD2 and POLD3 are found amplified in the vast majority of cancers. Hydroxyurea destabilizes Fe-S centers, which also compromises the stability of the DNA polymerase δ complex. Inhibitors of checkpoint kinases and reagents that compromise DNA polymerase δ activity may act synergistically to arrest growth in transformed cells.

Pseudomonas citronellolis is known to contain a pyruvate carboxylase with α4β4 composition. All the other pyruvate carboxylases investigated so far are made up of four seemingly identical subunits. Nevertheless, this exceptional pyruvate carboxylase exhibits a size and overall shape similar to other pyruvate carboxylases. Electron microscopic affinity labeling with avidin revealed that the prosthetic biotin groups (one per αβ unit, i.e. four per enzyme particle) are located close to the inter‐unit junctions of pairs of αβ units making up the enzyme. This position of the prosthetic biotin groups is very similar to the location of the biotin in the other carboxylases.